Marc Fajardo

ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein

Degradative fragments of cartilage oligomeric matrix protein (COMP) have been observed in arthritic patients. The physiological enzyme(s) that degrade COMP, however, remain unknown. We performed a yeast two‐hybrid screen (Y2H) to search for proteins that associate with COMP to identify an interaction partner that might degrade it. One screen using the epidermal growth factor (EGF) domain of COMP as bait led to the discovery of ADAMTS‐7. Rat ADAMTS‐7 is composed of 1595 amino acids, and this protein exhibits higher expression in the musculoskeletal tissues. COMP binds directly to ADAMTS‐7 in vitro and in native articular cartilage. ADAMTS‐7 selectively interacts with the EGF repeat domain but not with the other three functional domains of COMP, whereas the four C‐terminal TSP motifs of ADAMTS‐7 are required and sufficient for association with COMP. The recombinant catalytic domain and intact ADAMTS‐7 are capable of digesting COMP in vitro. The enzymatic activity of ADAMTS‐7 requires the presence of Zn2+ and appropriate pH (7.5–9.5), and the concentration of ADAMTS‐7 in cartilage and synovium of patients with rheumatoid arthritis is significantly increased as compared to normal cartilage and synovium. ADAMTS‐7 is the first metalloproteinase found to bind directly to and degrade COMP.—Liu, C., Kong, W., Ilalov, K., Yu, S., Xu, K., Prazak, L., Fajardo, M., Sehgal, B., Di Cesare, P. E. ADAMTS‐7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J. 20, E129–E140 (2006)

Disease-modifying therapies for osteoarthritis: Current status

Osteoarthritis, the most common form of arthritis, is a debilitating progressive disease principally affecting the elderly. Osteoarthritis therapy has evolved in the past few decades from symptomatic treatment to possible disease-modifying solutions. In this paper, the pathophysiology of osteoarthritis is first reviewed, including an examination of the mechanisms underlying osteoarthritis and discussions of the roles of cartilage, synovial fluid and subchondral bone. The remainder of the paper discusses therapeutic approaches in current use and those in development, with special attention given to pharmacological treatments. Current approaches to treating osteoarthritis — i.e. medications; nonpharmacological modalities, such as physical therapy, exercise, weight management and orthotics; and (as a last resort) surgery — focus on reducing pain and improving (or at least maintaining) mobility. Drugs currently used to treat osteoarthritis fall into several categories: analgesics, NSAIDs, cyclo-oxygenase-2 (COX-2) inhibitors, corticosteroids, viscosupplementation, and symptomatic slow-acting drugs (‘nutraceuticals’). The analgesics (paracetamol [acetaminophen] and opiates) have demonstrated less symptomatic efficacy than NSAIDs, while the latter have displayed mixed results in terms of joint space narrowing. COX-2 inhibitors have been demonstrated to be equal to or superior to NSAIDs in effectiveness. However, once considered a safer alternative, COX-2 inhibitors have become the subject of intense scrutiny since recent clinical evidence has cast suspicion on their cardiovascular safety profile. Injectable therapies, such as corticosteroids and viscosupplementation have elicited favorable short-term response but no long-term structural modification. On the other hand, the slow-acting drugs, especially chondroitin and glucosamine sulfate, have shown promising results. Also reviewed are other established and experimental therapies that seek to modify and/or even reverse the course of osteoarthritis. These include such medications as colchicine, bisphosphonates and hormones; dietary therapeutics, such as ginger extract and green tea; and such truly experimental treatments as matrix metalloproteinase inhibitors, cytokines, nitric oxide, growth factors and gene therapy. Osteoarthritis continues to be a difficult disorder to treat, as there is no cure as such and current treatments focus mainly on relieving pain and maintaining joint function. The search nevertheless continues for management regimens that can slow, alter or reverse the degenerative processes of osteoarthritis.

Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin

OBJECTIVE As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988-90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800-8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a(2)M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a(2)M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP. METHODS An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1-beta (IL-1beta) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a(2)M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a(2)M were also analyzed. RESULTS The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-alpha- or IL-1beta-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-alpha- or IL-1beta-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a(2)M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a(2)M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. CONCLUSION Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, a(2)M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a(2)M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.

Levels of Expression for BMP-7 and Several BMP Antagonists May Play an Integral Role in a Fracture Nonunion: A Pilot Study

Delays in bone healing or even the development of a nonunion could be related to the concentrations and/or functions of the bone morphogenetic proteins (BMPs). The RNA expression profile of the BMPs within fracture nonunion tissue is unknown. This preliminary descriptive study was performed to define the RNA profiles of the BMPs, their receptors, and their inhibitors within human fracture nonunion tissue and correlate them to matched healing bone. All patients had hypertrophic nonunions. Tissue samples taken from the nonunion site of 15 patients undergoing surgical treatment for an established nonunion were analyzed. The RNA expression patterns of BMP-2, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8; BMP receptor Types IA, IB, and II; and the BMP inhibitors chordin, Noggin, Drm (Gremlin), and follistatin were determined in the nonunion (fibrous tissue) and healing bone (callus tissue) using quantitative real-time PCR. Comparison between the nonunion and healing bone samples revealed substantially elevated concentrations of BMP-4, Drm/Gremlin, follistatin, and Noggin in nonunion tissue when compared to healing bone. In contrast, BMP-7 concentration was higher in the healing bone. Our data suggest inhibition of BMP-7, by Drm (Gremlin), follistatin, and Noggin and upregulation of BMP-4 may play an integral role in the development of nonunions.

Matrix metalloproteinases that associate with and cleave bone morphogenetic protein-2 in vitro are elevated in hypertrophic fracture nonunion tissue.

Objectives: A delayed union or a nonunion of a fracture is a potentially adverse complication. Understanding the mechanisms of nonunion development may lead to improved treatment modalities. Proteases such as the matrix metalloproteinases play important roles in bone remodeling and repair, in which an imbalance or a nonfunctioning enzyme may lead to defects in bone healing (nonunion). The purpose of this pilot study was twofold: first to define an mRNA expression profile of all the matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases with thrombospondin motif (ADAMTS) enzymes, and their inhibitors (TIMPs) within fracture nonunion tissue, and second to compare this profile with mineralized fracture callus. Methods: Using a systematic real-time polymerase chain reaction, we screened the gene expression profiles of all members of the MMPs, ADAMTS, and their inhibitor TIMPs on human fracture nonunion tissue and matched mineralized callus tissue. Significant results were further analyzed using Western immunoblotting, immunohistochemistry, and in vitro protein interaction assays with bone morphogenetic protein-2. Results: This analysis confirmed MMP-7 and MMP-12 as two unidentified enzymes expressed in fracture nonunion tissue. Both MMP-7 and MMP-12 mRNAs were significantly elevated in nonunion tissue when compared with local mineralized callus from the same site (P < 0.001); the elevated protein levels of interest were visualized through immunoblotting and immunohistochemistry. In addition, these two MMPs were found to directly bind to and degrade bone morphogenetic protein-2 in vitro. Conclusion: Collectively, our findings indicate that tissue present at the site of hypertrophic nonunions commonly expresses significantly higher levels of MMP-7 and MMP-12 in relation to mineralized fracture callus. Both were found to directly inactivate bone morphogenetic protein-2 in vitro, the best established growth factor in bone formation and repair.

Effect of a Perioperative Intra-articular Injection on Pain Control and Early Range of Motion Following Bilateral TKA

Pain control after total knee arthroplasty (TKA) is integral in the immediate postoperative period for early rehabilitation. Numerous different methods of postoperative analgesia are available, but each has its own risk of adverse side effects. This study was performed to prospectively evaluate the benefits of an intra-articular analgesic injection in patients undergoing bilateral TKA.Thirty consecutive patients undergoing bilateral TKA were enrolled in this prospective, randomized, controlled study. Each patient was randomized to receive (1) a perioperative intra-articular mixture of morphine, bupivacaine with epinephrine, and ketorolac in 1 knee, and (2) injectable sterile saline in the contralateral knee. Each patient acted as his or her own internal control. The pharmacologically injected knee had statistically significantly less pain immediately postoperatively when compared to the control knee and displayed significantly increased range of motion within the first week of rehabilitation.The use of an intraoperative intra-articular injection with the above drug combination significantly reduces patient pain and increases postoperative mobility with no apparent risks following bilateral TKA.

Quantitative assessment of the bone morphogenetic protein expression from alternate bone graft harvesting sites.

Objective: Bone morphogenetic proteins (BMPs) play important roles in the stimulation of osteogenesis and osteoinduction during bone fracture healing and their expression levels may be important for bone graft efficacy. The objective of this study was to determine if there are variations in the expression of BMPs and their receptors in various bone graft harvesting sites. We analyzed autogenous marrow aspirates obtained from three different graft sites for the mRNA levels of BMPs and their receptors. Methods: Using real-time polymerase chain reaction, we analyzed the mRNA levels of BMPs and their receptors in autogenous bone marrow aspirates obtained from three different bone graft sites of 10 different human subjects. Collection of autogenous bone marrow from the iliac crest, the proximal humerus, and the proximal tibia was performed using standard sterile techniques in the operating room as part of surgery to treat an established fracture nonunion. Results: The mRNA levels of BMP-2 and BMP-5 were the highest in the bone marrow aspirates from the three different sites, whereas the mRNA levels of the other osteoinductive BMPs (BMP-4, -5, -6, -7, -8, and -9) were lower. The mRNA levels of BMP-3, an inhibitor of osteogenesis, were the lowest in the bone marrow aspirates of all three different sites. There were no statistical significant differences in the mRNA levels of any of the BMPs or their receptors investigated in this study in the bone marrow of the three different sites. Conclusion: Because no statistical significant differences in the mRNA levels of the BMPs and their receptors were detected in the bone marrow aspirates from the three different sites, our findings suggest that potential differences of various graft sites in the augmentation of bone healing does not result from different expression levels of BMPs.

Reoperation for failed shoulder reconstruction following brachial plexus birth injury

Background Various approaches have been developed to treat the progressive shoulder deformity in patients with brachial plexus birth palsy. Reconstructive surgery for this condition consists of complex procedures with a risk for failure. Case presentations This is a retrospective case review of the outcome in eight cases referred to us for reoperation for failed shoulder reconstructions. In each case, we describe the initial attempt(s) at surgical correction, the underlying causes of failure, and the procedures performed to rectify the problem. Results were assessed using pre- and post-operative Mallet shoulder scores. All eight patients realized improvement in shoulder function from reoperation. Conclusions This case review identifies several aspects of reconstructive shoulder surgery for brachial plexus birth injury that may cause failure of the index procedure(s) and outlines critical steps in the evaluation and execution of shoulder reconstruction.